ANTISENSE OLIGONUCLEOTIDES AGAINST cPLA2, COMPOSITIONS AND USES THEREOF

ABSTRACT

Antisense oligonucleotides against cPLA 2  are provided, which are capable of inhibiting cPLA 2  expression as well as superoxide production, especially in phagocytes. These antisense oligonucleotides are powerful agents for the treatment of inflammatory conditions, in particular arthritis, as well as in neurodegenerative diseases. The antisense oligonucleotides or compositions comprising the same may be used in methods of treatment of such diseases.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/547,113 filed on Jul. 12, 2012, which is a continuation of U.S.patent application Ser. No. 11/568,169 filed on Aug. 19, 2007, now U.S.Pat. No. 8,242,255, which is a National Phase of PCT Patent ApplicationNo. PCT/IL2005/000399 having International filing date of Apr. 17, 2005,which claims the benefit of priority of Israel Patent Application No.161579 filed on Apr. 22, 2004, now abandoned. The contents of the aboveapplications are all incorporated herein by reference as if fully setforth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 58468SequenceListing.txt, created on Feb. 12,2014, comprising 6,409 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of use of antisenseoligonucleotides in the treatment of medical conditions. Morespecifically, the present invention describes novel antisenseoligonucleotides for inhibition of phospholipase A₂ (PLA₂) and treatmentof conditions associated with the activation of this molecule.

BACKGROUND OF THE INVENTION

All publications mentioned throughout this application are fullyincorporated herein by reference, including all references citedtherein.

Inflammation is the body's response to injury, infection or to moleculesperceived by the immune system as foreign. Absent, excessive oruncontrolled inflammation results in a vast array of diseases such asasthma, arthritis and autoimmune diseases, adult respiratory distresssyndrome (ARDS), cardiovascular inflammation and gastrointestinalinflammation. Numerous studies have demonstrated the participation ofprimed neutrophils, monocytes and macrophages in such inflammatorydiseases. More recently, the role of superoxides release by microgliacells in the pathogenesis of neurodegenerative diseases such asAlzheimer's disease (AD), Parkinson's disease (PD) and amyotrophiclateral sclerosis (ALS) as well as brain ischemic and traumatic injuryhas also been documented.

The production of superoxides by the phagocyte NADPH oxidase andpro-inflammatory lipid mediators by phospholipase A₂ are among the mostimportant functions for host defense. However, during alteredphysiological states, superoxides and lipid mediators promoteinflammatory reactions and participate in processes that lead to tissueinjury and the pathophysiology of various inflammatory diseases.Nowadays, non-steroidal anti-inflammatory drugs (NSAIDs) are one of themost widely prescribed drugs for the treatment of inflammatoryconditions. However, they present unwanted side effects, the most commonbeing ulceration and bleeding in the gastrointestinal tract. Moreover,these drugs reduce only the production of prostaglandins and do notaffect the production of leukotrienes which have a pivotal role in therecruitment of neutrophils to the site of inflammation. Thus, the searchfor new anti-inflammatory drugs with fewer side effects continues.Numerous trials have been conducted with agents that block theinflammatory cascade, like corticosteroids, antiendotoxin antibodies,TNF antagonists, IL-1 receptor antagonists and other agents, withoutsignificant success.

The present inventor has developed a cell line, stable clones of PLB-985cells lacking the expression of cytosolic phospholipase A₂ (cPLA₂), anddemonstrated that cPLA₂, in addition to its known role in the productionof pro-inflammatory lipid mediators, is essential for activation of thephagocyte NADPH oxidase complex after its assembly. The associationbetween these two enzymes provides the molecular basis for activation ofthe assembled NADPH oxidase by arachidonic acid (AA) released by cPLA₂[Dana, R. et al. (1998) J. Biol. Chem. 273:441-5; Lowenthal, A. andLevy, R. (1999) J. Biol. Chem. 274: 21603-10; Levy, R. et al. (2000)Blood. 95:660-5; Pessach, I. et al. (2001) J. Biol. Chem. 276:33495-503;Shmelzer, Z. et al. (2003) J. Cell Biol. 162:683-692; Tarsi-Tsuk, D. andLevy, R. (1990) J. Immunol.; 144:2665-2670; Dana, R. et al. (1994)Biochem J. 297:217-223; Hazan-Halevy, I. et al. (2000) J. Biol. Chem.275:12416-12423]. Since cPLA₂ is required for oxidase activation, itsinhibition should not only diminish the formation of inflammatorymediators, but should also regulate the uncontrolled accelerated releaseof oxygen radicals that participate in the pathogenesis of inflammatorydiseases. Moreover, the inventor's studies have shown that duringinflammation in vivo or inflammatory conditions in vitro, the level andactivity of both cPLA₂ and NADPH oxidase enzymes are elevated inneutrophils and monocytes [Levy, R. et al. (1994) Biochim. Biophys. Acta1220:261-265; Shaked, G. et al. (1994) J. Trauma 37:22-29; Levy, R. etal. (2000) Blood 95:660-665; Levy, R. and Malech, H. (1991) J. Immunol.147:3066-3071; Levy, R. et al. (1994) Biochim. Biophys. Acta1220:253-260; Reizenberg, K. et al. (1997) Eur. J. Clin. Invest.27:398-404]. Surprisingly, a recent report described that addition ofcPLA₂ inhibitor pyrrolidine to neutrophils did not inhibit NADPH oxidaseactivity [Rubin, B. B. et al. (2005) J. Biol. Chem. 280:7519-29],however this effect might have been due to the methodology applied,which did not allow sufficient accumulation of the drug in theneutrophils (data not shown). Although methods of treating inflammatoryconditions by inhibiting cPLA₂ have been described, they involved theuse of substances like trifuoromethylketone (TFMK), causingdose-dependent attenuation of airway inflammation [US Patent ApplicationNo. 20020165119, USSN 062730], or indole compounds, which inhibitedvarious forms of PLA₂ [U.S. Pat. No. 6,797,708], but no inhibitor uniqueto cPLA₂ has been described for treatment of inflammation to date.Currently, potent cytosolic PLA₂ inhibitors are not available forclinical use in human or animals. All inhibitors against cPLA₂ so farwere engineered to compete with the substrate. Since all types of PLA₂cleave the fatty acid from the sn-2 position of phospholipids, they arealso inhibited by the same inhibitors (although some times with lowerefficiency). Although several compounds were described as specificinhibitors of cPLA₂, they were found to also inhibit other PLA₂ enzymesand vice versa. Because of the lack of specific inhibitor for each PLA₂subtype, the antisense technology provides an effective approach toinhibit a specific type of PLA₂. Indeed, the results presented hereinsuggest that a drug targeted directly to cPLA₂ will specifically inhibitcPLA₂ activity. Moreover it also results in the regulation of both cPLA₂and NADPH oxidase to produce pro-inflammatory mediators and superoxides.

Antisense oligonucleotides targeted against the cPLA₂ mRNA sequence havebeen reported in the past as capable of inhibiting cPLA₂ transcriptexpression [U.S. Pat. No. 6,008,344]. However, these oligonucleotidesdid not demonstrate inhibition of cPLA₂ protein expression, and wereintroduced into cells in the presence of lipofectin.

In addition, three other antisense oligonucleotides targeted to cPLA₂have been described: P1 (Table 1, SEQ. ID. No.8) [Roshak, A. (1994) J.Biol. Chem. 269(42): 25999-26005; Muthalif, M. M. et al. (1996) J. Biol.Chem. 271(47): 30149-30157; Marshall, L. (1997) J. Biol. Chem. 272(2):759-765; Anderson, K. M. et al. (1997) J. Biol. Chem. 272(48):30504-30511]; P2 (Table 1, SEQ. ID. No.9) [Li, Q. and Cathcart, M. K.(1997) J. Biol. Chem. 272(4): 2404-2411; Zhao, X. et al. (2002) J. Biol.Chem. 277(28): 25385-25392]; and P3 (5′-GTGCTGGTAAGGATCTAT-3′; SEQ. ID.No.12) [Locati, M. (1996) J. Biol. Chem. 271(11): 6010-6016], mainlyevaluating the effect of inhibiting cPLA₂ in smooth muscle cells andhuman monocytes function. P1 was used together with lipofectin. P1 andP2, with phosphorothioate modifications in all bases, had a significanteffect only when used at 5 μM, which the present inventor found to betoxic to the cells. P3 was used at 10 μM (or even higher concentration,10 times higher than what was used by the present inventor).

Thus, it is an object of the present invention to provide novelantisense oligonucleotides against the cPLA₂ mRNA, and their use in theinhibition of cPLA₂ expression and superoxide production, in order toinhibit pro-inflammatory processes. Consequently, the antisenseoligonucleotides claimed in the present invention are also sought asanti-inflammatory agents.

Other uses and objects of the invention will become clear as thedescription proceeds.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides antisenseoligonucleotides directed against the open reading frame (ORF) of thecytosolic phospholipase A₂ (cPLA₂) mRNA sequence, and functionalanalogs, derivatives or fragments thereof, wherein the complementarityof said antisense oligonucleotide is within the region betweennucleotides 145 to 400 of said ORF, and wherein said antisenseoligonucleotide is capable of inhibiting the expression of the cPLA₂protein.

In one embodiment, the antisense oligonucleotide of the invention isfrom 15 up to 30 nucleotides long, preferably 17 to 21 nucleotides long.

Said antisense oligonucleotide directed against the 5′ region of theopen reading frame of the cPLA₂ mRNA sequence has the sequence asdenoted by any one of SEQ. ID. No.1, SEQ. ID. No.2, SEQ. ID. No.3, SEQ.ID. No.4, SEQ. ID. No.5, and SEQ. ID. No.6, and as detailed in Table 1.

The antisense oligonucleotides of the invention can be chemicallymodified, so as to possess improved endonuclease resistance.

Thus, in another embodiment of the antisense oligonucleotide of theinvention, a phosphorothioate modification may be present on the firstthree and/or the last three nucleotides of said oligonucleotides. Inaddition, another phosphorothioate modification may be found on thetenth nucleotide of said oligonucleotide, as for example in theoligonucleotides denoted by SEQ. ID. Nos. 4 and 5.

In a further embodiment of the antisense oligonucleotide of theinvention, further modifications, like 2-O-methylation, may be found inthe first three and/or the last three nucleotides of saidoligonucleotide.

As a result of the properties presented in the present study, theantisense oligonucleotide may be used as an inhibitor of inflammationprocesses related to cPLA₂ expression.

Therefore, the antisense oligonucleotide of the invention is for use inthe treatment and/or prevention of any one of rheumatoid arthritis,adult respiratory distress syndrome (ARDS), asthma, rhinitis, idiopathicpulmonary fibrosis, peritonitis, cardiovascular inflammation, myocardialischemia, reperfusion injury, atherosclerosis, sepsis, trauma, diabetestype II, retinopathy, psoriasis, gastrointestinal inflammation,cirrhosis and inflammatory bowel disease, and neurodegenerativediseases, such as for example Alzheimer's disease (AD), Parkinson'sdisease (PD), amyotrophic lateral sclerosis (ALS), as well as brainischemic and traumatic injury, i.e. in all diseases where oxidativestress has a significant role in its pathogenesis, and in which there isaccelerated release of eicosanoids and superoxides by reactivemicroglia.

Thus, the antisense oligonucleotide of the invention may be used forinhibiting superoxide production and release. In particular, saidinhibition is effectuated in neutrophils, monocytes and macrophages,preferably in neutrophils.

Optionally the antisense oligonucleotide of the invention may be labeledwith one of fluorescent, radioactive, metal particle, and any suitablelabeling means.

In a second aspect, the present invention relates to a pharmaceuticalcomposition comprising as active agent at least one antisenseoligonucleotide as defined in the invention, or functional analogs,derivatives or fragments thereof.

Thus, the antisense oligonucleotide of the invention is generallyprovided in the form of pharmaceutical compositions. Said compositionsare for use by injection, topical administration, or oral uptake.

Alternatively, the pharmaceutical composition of the invention maycomprise as active agent a combination of at least two antisenseoligonucleotides as defined in the invention, or functional analogs,derivatives or fragments thereof. Preferably, said combination comprisesthe following oligonucleotides: SEQ. ID. No.1 together with SEQ. ID.No.3, or SEQ. ID. No.1 together with SEQ. ID. No.2, or SEQ. ID. No.1together with SEQ. ID. No.6, or SEQ. ID. No.1 together with SEQ. ID.No.2 and SEQ. ID. No.3, or SEQ. ID. No.4 together with SEQ. ID. No.6, orSEQ. ID. No.2 together with SEQ. ID. No.6, or SEQ. ID. No.2 togetherwith SEQ. ID. No.3, or SEQ. ID. No.3 together with SEQ. ID. No.6.

The pharmaceutical composition of the invention is intended for medicaluse.

In one embodiment, the pharmaceutical composition of the invention isintended for the treatment of inflammation processes related to cPLA₂expression and/or free radical release by phagocyte NADPH oxidase.

In another embodiment, the pharmaceutical composition of the inventionis intended for the treatment of inflammatory conditions, wherein saidinflammatory conditions may be any one of rheumatoid arthritis, ARDS,asthma, rhinitis, idiopathic pulmonary fibrosis, peritonitis,cardiovascular inflammation, myocardial ischemia, reperfusion injury,atherosclerosis, sepsis, trauma, diabetes type II, retinopathy,psoriasis, gastrointestinal inflammation, cirrhosis and inflammatorybowel disease, CNS-related diseases such as the neurodegenerativediseases AD, PD, ALS, as well as brain ischemic and traumatic injury,i.e. in all diseases where oxidative stress has a significant role inits pathogenesis, and in which there is accelerated release ofeicosanoids and superoxides by reactive microglia.

In a further embodiment, the pharmaceutical composition of the inventionis intended for the treatment of conditions related to Aβ plaqueaccumulation. Said conditions are generally CNS-related diseases,particularly the neurodegenerative diseases Alzheimer's, Parkinson's andALS, or brain ischemic and traumatic head injury.

The pharmaceutical composition of the invention may optionally furthercomprise buffers, additives, stabilizers, diluents and/or excipients.

In another aspect, the present invention provides the use of theantisense oligonucleotide as defined in the invention, for thepreparation of a pharmaceutical composition for the treatment and/orprevention of inflammatory conditions, wherein said inflammatoryconditions may be any one of rheumatoid arthritis, ARDS, asthma,rhinitis, idiopathic pulmonary fibrosis, peritonitis, cardiovascularinflammation, myocardial ischemia, reperfusion injury, atherosclerosis,sepsis, trauma, diabetes type II, retinopathy, psoriasis,gastrointestinal inflammation, cirrhosis and inflammatory bowel disease,neurodegenerative diseases such as AD, PD and ALS, as well as brainischemic and traumatic injury, i.e. in all diseases where oxidativestress has a significant role in its pathogenesis, and in which there isaccelerated release of eicosanoids and superoxides by reactivemicroglia.

In a further aspect, the present invention provides the use of anantisense oligonucleotide as defined in the invention for the treatmentof conditions associated with cPLA₂ activation.

In addition, the present invention presents the use of an antisenseoligonucleotide as defined in the invention, for the treatment and/orprevention of conditions related to Aβ plaque accumulation. Generallysaid conditions are CNS-related diseases selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, ALS, brainischemic injury and traumatic head injury.

In an even further aspect, the present invention presents a method oftreatment of conditions associated with cPLA₂ activation, comprisingadministering a therapeutically effective amount of at least oneantisense oligonucleotide as defined in the invention, or compositionscomprising thereof, to a subject in need.

Therefore, in yet another aspect, the present invention provides amethod of treatment of inflammatory conditions, wherein saidinflammatory conditions are any one of rheumatoid arthritis, ARDS,asthma, rhinitis, idiopathic pulmonary fibrosis, peritonitis,cardiovascular inflammation, myocardial ischemia, reperfusion injury,atherosclerosis, sepsis, trauma, diabetes type II, retinopathy,psoriasis, gastrointestinal inflammation, cirrhosis and inflammatorybowel disease, neurodegenerative diseases such as AD, PD and ALS, aswell as brain ischemic and traumatic injury, i.e. in all diseases whereoxidative stress has a significant role in its pathogenesis, and inwhich there is accelerated release of eicosanoids and superoxides byreactive microglia, comprising administering a therapeutically effectiveamount of at least one antisense oligonucleotide as defined in theinvention, or compositions comprising thereof, to a subject in need.

In one more aspect, the present invention provides a method of treatmentof conditions related to Aβ plaque accumulation, comprisingadministering a therapeutically effective amount of at least oneantisense oligonucleotide as defined in the invention, or compositionscomprising thereof, to a subject in need. Said conditions are generallyneurodegenerative diseases, particularly Alzheimer's and Parkinson'sdisease.

Finally, the present invention provides an in vivo, ex vivo or in vitromethod of inhibiting cPLA₂ expression and/or activity, comprisingcontacting cells, preferably phagocytes, i.e. neutrophils, monocytes,macrophages and/or microglia, with the antisense oligonucleotidedescribed in the invention or with compositions comprising thereof, fora suitable amount of time. These antisense oligonucleotides inhibitedthe cPLA₂ activity in fibroblasts, neuronal cells and endothelial cells,but with lower efficiency compared to phagocytes, probably due to lowerpermeability of the former cells to the antisense oligonucleotides (datanot shown).

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: cDNA Sequence of cPLA₂ (SEQ. ID. No. 7), with position ofantisense oligonucleotides C2, C3, C4, C8, C9 and C10 highlighted.

C2 encompasses nucleotides 347-366; C3 encompasses nucleotides 155-174;C4 encompasses nucleotides 379-399; C8 encompasses nucleotides 330-346;C9 encompasses nucleotides 183-202; and C10 encompasses nucleotides290-306.

FIGS. 2A-2B: Comparison between two anti-cPLA₂ antibodies (Ab):

FIG. 2A: Levy's Ab [Hazan et al. (1997) id ibid.]

FIG. 2B: commercial antibodies.

Dil.=dilution.

FIG. 3A-3B: Effect of cPLA₂ antisense PPS oligonucleotides on cPLA₂expression and superoxide production in human peripheral bloodmonocytes.

FIG. 3A: Western blot analysis showing inhibition of cPLA₂ expressiondetected by anti-cPLA₂ antibodies after treatment with antisense PPSoligonucleotides C2, C3, C4, C9, and the combination of C2+C4 (2+4), incomparison to control and to the antisense P1 [Roshak, A et al. (1994)id ibid.; Marshall, L. et al. (1997) id ibid.; Muthalif, M. M. et al.(1996) id ibid.; Anderson, K. M. et al. (1997) id ibid.] and P2 [Li, Q.and Cathcart, M. K. (1997) id ibid.]. The level of cPLA₂ in thedifferent treatment was quantified by densitometry and is presentedbelow the Western blot analysis. Lev.=levels; dens.=densitometry.

FIG. 3B: Histogram showing inhibition of superoxide production (SOprod.) by cPLA₂ antisense PPS oligonucleotides C2, C3, C4, C9, and thecombination of C2+C4 (2+4), in comparison to control and to theantisense P1 [Muthalif, M. M. et al. (1996) id ibid.; Anderson, K. M. etal. (1997) id ibid.] and P2 [Li, Q. and Cathcart, M. K. (1997) idibid.].

It is important to note the higher efficiency of the antisense PPSoligonucleotides of the invention (denoted as 2, 3, 4 and 9) versus theantisense oligonucleotides P1 and P2 described previously.

FIG. 4A-4B: Effect of cPLA₂ antisense PPS oligonucleotides on cPLA₂expression and superoxide production in peripheral blood humanmonocytes.

FIG. 4A: Western blot analysis showing inhibition of cPLA₂ expressiondetected by anti-cPLA₂ antibodies after treatment with antisensepartially phosphorothioated (PPS) oligonucleotides C2, C8, C9, C10 andthe combinations C3+C10 and C8+C10, in comparison to control and to theoligonucleotides IS1 and IS2 [U.S. Pat. No. 6,008,344]. The level ofcPLA₂ in the different treatment was quantified by densitometry and ispresented below the Western blot analysis. Lev.=levels;dens.=densitometry.

FIG. 4B: Histogram showing inhibition of superoxide production (SOprod.) by cPLA₂ antisense PPS oligonucleotides C2, C8, C9, C10 and thecombinations C3+C10 and C8+C10, in comparison to control (cont.) and tothe oligonucleotides IS1 and IS2 [U.S. Pat. No. 6,008,344].

It is important to note the higher efficiency of the antisenseoligonucleotides of the invention (denoted as 2, 8, 9, and 10 in theFigure) versus the antisense oligonucleotides described in U.S. Pat. No.6,008,344 (IS1 and IS2).

FIG. 5A-5C: Effect of cPLA₂ antisense PPS oligonucleotides on cPLA₂expression and superoxide production in peritoneal murine macrophages.

FIG. 5A: Western blot analysis showing inhibition of cPLA₂ expressiondetected by anti-cPLA₂ antibodies after treatment with antisense PPSoligonucleotides C3 and C4 and the combination C3+C4.

FIG. 5B: Histogram showing inhibition of superoxide production by cPLA₂antisense PPS oligonucleotides C3 and C4 and the combination C3+C4.

FIG. 5C: Histogram showing inhibition of superoxide production by cPLA₂antisense PPS oligonucleotides C2, C3, C4, C10 and the combinationsC2+C10, C3+C10, C4+C10 and C3+C4.

Abbreviations: cont., control; SO gen., superoxide generation.

FIG. 6: Correlation between inhibition of cPLA₂ expression (exp.) andsuperoxide production (SO prod.) following treatment with cPLA₂antisense PPS oligonucleotides. Cont.=control.

FIG. 7A-7C: Effect of cPLA₂ antisense PPS oligonucleotides on cPLA₂expression and superoxide production in peripheral blood humanneutrophils.

FIG. 7A: Western blot analysis showing inhibition of cPLA₂ expressiondetected by anti-cPLA₂ antibodies after treatment with antisense PPSoligonucleotides C2 or C4 and the combination C2+C4. The level of cPLA₂in the different treatment was quantified by densitometry and ispresented below the Western blot analysis.

FIG. 7B: Histogram showing inhibition of superoxide production by cPLA₂antisense oligonucleotides C2, C4 or C10 and the combinations C2+C4,C2+C10, C4+C10, C2+C4+C10 following PMA treatment.

FIG. 7C: Histogram showing inhibition of superoxide production by cPLA₂antisense oligonucleotides C2, C4 or C10 and the combinations C2+C4,C2+C10, C4+C10, C2+C4+C10 following OZ treatment.

Abbreviations: Lev., levels; dens., densitometry; cont., control; SOgen., superoxide generation.

FIG. 8A-F: Inhibition of superoxide production stimulated by physiologicagonists following cPLA₂ antisense PPS oligonucleotides treatment inneutrophils and macrophages.

FIG. 8A: Histogram showing inhibition of superoxide production inPMA-stimulated neutrophils following 6 hours of incubation with thecPLA₂ antisense PPS oligonucleotides C3 or C4.

FIG. 8B: Histogram showing inhibition of superoxide production infMLP-stimulated neutrophils following 4 hours of incubation with thecPLA₂ antisense PPS oligonucleotides C3 or C4.

FIG. 8C: Histogram showing inhibition of superoxide production inOZ-stimulated neutrophils following 4 hours of incubation with the cPLA₂antisense PPS oligonucleotides C3 or C4.

FIG. 8D: Histogram showing inhibition of superoxide production inPMA-stimulated monocytes following 16 hours of incubation with the cPLA₂antisense PPS oligonucleotides C3 or C4.

FIG. 8E: Histogram showing inhibition of superoxide production infMLP-stimulated monocytes following 16 hours of incubation with thecPLA₂ antisense PPS oligonucleotides C3 or C4.

FIG. 8F: Histogram showing inhibition of superoxide production inOZ-stimulated monocytes following 16 hours of incubation with the cPLA₂antisense PPS oligonucleotides C3 or C4.

Abbreviations: SO prod., superoxide production; Act., activity.

FIG. 9A-9D: Inhibition of stimulated superoxide production in ratmicroglia by cPLA₂ antisense PPS oligonucleotides.

FIG. 9A: Western blot analysis showing inhibition of cPLA₂ expressiondetected by anti-cPLA₂ antibodies after treatment with antisense PPSoligonucleotides C2 or C4.

FIG. 9B: Graph demonstrating the kinetics of the inhibition ofsuperoxide production in PMA-activated rat microglia cells followingtreatment with the cPLA₂ antisense oligonucleotides C2, C4, C8 or C10,and the combinations C2+C10 or C4+C10.

FIG. 9C: Histogram showing the effect of cPLA₂ antisense PPSoligonucleotides (C2, C4, C8 and C10, and the combinations C2+C10 andC4+C10) on superoxide production in rat microglia cells following PMAstimulation.

FIG. 9D: Histogram showing the effect of cPLA₂ antisense PPSoligonucleotides (C2, C4, C8 and C10, and the combinations C2+C10 andC2+C4) on superoxide production in rat microglia cells following Amyloidβ stimulation.

Abbreviations: rest., resting; act., activated; as, antisense; kin.,kinetics; T., time, min., minutes.

FIG. 10A-10C: Animal model of collagen-induced arthritis (CIA)

FIG. 10A: A picture of exacerbated CIA in experimental compared tocontrol (cont.) mice.

FIG. 10B: Histological assessment of representative section of jointhistopathology on whole paws of CIA mice compared to control (cont.)(X100).

FIG. 10C: Infiltration of inflammatory cells (X400) in CIA mice.

FIG. 11A-11B: Treatment with a combination of 3 cPLA₂ antisense PPSoligonucleotides (“Cocktail”) caused remission of arthritis.

FIG. 11A: Photograph of swollen limb of CIA mouse (top, art.=arthritis)and limb CIA mouse post-treatment with the cocktail (bottom,as=antisense treatment). Both photographs were taken at samemagnification and same distance from the animal.

FIG. 11B: Reduction in disease severity score (dis. sev. sc.), in serum(ser.) IL-6 and in serum TNFα after i.v. treatment with the cocktail(mean±SEM, from 5 mice in each group).

FIG. 12A-12B: Number and composition of peritoneal cell population afterinduction of sterile peritonitis.

FIG. 12A: Changes in peritoneal cell count in sterile peritonitis mice(mean±SEM, from 6 mice in each group).

FIG. 12B: Changes in the composition of the peritoneal cell populationin sterile peritonitis mice.

Abbreviations: ce. no., cell number; T., time; h, hours; neu,neutrophils; mac, macrophages; lymp, lymphocytes.

FIG. 13A-13B: LTB₄ levels after induction of sterile peritonitis.

FIG. 13A: LTB₄ levels in the serum (ser.).

FIG. 13B: LTB₄ levels in the peritoneal cavity (per. cav.).

Mean±SEM from 5 mice in each group. T., time; h, hours.

FIG. 14A-14C: The effect of antisense PPS oligonucleotide cocktailtreatment on peritoneal cell population and activity after 24 hours ofinduction.

FIG. 14A: FACS analysis of cell population (ce. pop.) composition.Neu=neutrophils.

FIG. 14B: Graph demonstrating cell count (ce. co.).

FIG. 14C: Superoxide production (SO prod.) by stimulated peritonealcells of control healthy mice (H), of sterile peritonitis mice (P) andof sterile peritonitis mice after 24 h of cocktail i.v. injection(P+AS).

FIG. 15A-15B: cPLA₂ antisense PPS oligonucleotide cocktail treatmentdecreased neutrophils number and superoxide production in inflammationsite 24 hours after peritonitis induction.

FIG. 15A: Graph demonstrating percentage of neutrophils (neu) with andwithout antisense treatment.

FIG. 15B: Graph presenting stimulated superoxide production (SO prod.)by PMA of peritoneal cells isolated from sterile peritonitis micewithout and with antisense treatment.

FIG. 16: cPLA₂ antisense PPS oligonucleotide cocktail treatmentdecreased unstimulated superoxide production by resting peritoneal cellsin inflammation site 24 hours after peritonitis induction.

Shown is an example of detection of superoxide production by restingperitoneal cells isolated from sterile peritonitis mice assessed with 1μM Dihydrorhodamine-123, without and with antisense treatment.Co.=counts.

FIG. 17A-17B: Effect of cPLA₂ antisense PPS oligonucleotide cocktailtreatment on the peritoneal cell composition in mice with sterileperitonitis.

FIG. 17A: Peritoneal cell composition during sterile peritonitis.

FIG. 17B: Peritoneal cell composition post antisense treatment.

Abbreviations: ce. no., cell number; T., time; h, hours; neu,neutrophils; mac, macrophages; lymp, lymphocytes.

Abbreviations: Neu, neutrophils; mac, macrophages; lymp, lymphocytes;(means from 5 mice in each group).

FIG. 18A-18B: Effect of cPLA₂ antisense PPS oligonucleotide treatment onstimulated superoxide production (SO prod.) by peritoneal cells duringsterile peritonitis.

FIG. 18A: Stimulated superoxide production by peritoneal cells in micewith sterile peritonitis.

FIG. 18B: Stimulated superoxide production by peritoneal cells in micewith sterile peritonitis and treated with antisense oligonucleotides.

Mean±SEM from 5 mice in each group. T.=time; h=hours.

FIG. 19A-19B: cPLA₂ antisense PPS oligonucleotide cocktail treatmentdecreased peritoneal LTB4 levels.

FIG. 19A: LTB₄ levels in the peritoneum of sterile peritonitis mice.

FIG. 19B: LTB₄ levels in the peritoneum of sterile peritonitis micetreated with the cocktail.

Mean±SEM from 5 mice in each group. T.=time; h=hours.

FIG. 20A-20B: cPLA₂ antisense PPS oligonucleotide cocktail treatmentdecreased peritoneal neutrophils count.

FIG. 20A: Number of neutrophils (Neu No) in the peritoneum of sterileperitonitis mice.

FIG. 20B: Number of neutrophils (Neu No) in the peritoneum of sterileperitonitis mice treated with the cocktail.

Mean±SEM from 5 mice in each group. T.=time; h=hours.

FIG. 21A-21B: Accumulation of antisense oligonucleotides in peritonealblood cells 24 hours after injection.

FIG. 21A: Peritoneal blood cells, untreated.

FIG. 21B: Peritoneal blood cells treated with the antisense (as)oligonucleotides.

Upper panel: light microscope. Lower panel: confocal microscopy.

DETAILED DESCRIPTION OF THE INVENTION

In the present study, the inventors engineered six different antisenseoligonucleotides against cytosolic phospholipase A₂ (cPLA₂) mRNA. Asshown in the following Examples, each antisense by itself issignificantly potent in inhibiting the expression of cPLA₂, whiledifferent combinations of the oligonucleotides totally inhibited cPLA₂expression in the different phagocytic, as well as microglia cells fromhumans, mice and rats. Moreover, there is a striking correlation betweenthe inhibition of cPLA₂ expression by the antisense oligonucleotides andthe inhibition of superoxide production by NADPH oxidase, which has notbeen previously demonstrated.

Thus, the present invention provides antisense oligonucleotides directedagainst the open reading frame (ORF) of the cPLA₂ mRNA sequence(indicated in FIG. 1), and functional analogs, derivatives or fragmentsthereof, wherein the complementarity of said antisense oligonucleotideis within the region between nucleotides 145 to 400 of said ORF, andwherein said antisense oligonucleotide is capable of inhibiting theexpression of the cPLA₂ protein.

As shown in the following Examples, the antisense oligonucleotidesprovided by the invention can also inhibit superoxide production byinhibiting NADPH oxidase activity.

As referred to herein, SEQ. ID. No.7 relates to the cDNA sequencecorresponding to the cPLA₂ mRNA sequence [GenBank No. M68874].

Said region between nucleotides 145 to 400 of the cPLA₂ mRNA isparticularly useful for targeting, since it is much more efficient toprevent than to halt protein synthesis, once the process has alreadybegun (the latter being the strategy used in U.S. Pat. No. 6,008,344,for most of its cPLA₂ antisense sequences). Therefore, the antisenseoligonucleotides were designed as to target the region of translationsite (beginning of the ORF).

Nonetheless, it is important to mention that antisense targeting isstill very empirical, and a lot of experimentation is needed to find thespecific sequence and the optimum conditions for most effectivetargeting. As described herein for example, the present inventorsoriginally designed fourteen antisense oligonucleotides targeting theregion between nucleotides 145-400 of the cPLA₂ sequence, correspondingto the beginning of the ORF, but only six of those worked efficiently,and were then studied in more detail.

Said antisense oligonucleotide directed against the 5′ region of theopen reading frame of the cPLA₂ mRNA sequence has the sequence asdenoted by any one of SEQ. ID. No.1, SEQ. ID. No.2, SEQ. ID. No.3, SEQ.ID. No.4, SEQ. ID. No.5, and SEQ. ID. No.6, which sequences are detailedin Table 1.

As mentioned, the antisense oligonucleotides of the invention can bechemically modified, so as to possess improved endonuclease resistance.Any chemical modification which confers resistance towardsendonucleases, such as, but not limited to phosphorothioation or2-O-methylation, may be adopted.

Thus, a phosphorothioate modification may be present on the first threeand/or the last three nucleotides of the oligonucleotides of theinvention. In addition, another phosphorothioate modification may befound on the tenth nucleotide of said oligonucleotide, an innerpyrimidine, as for example in the oligonucleotides denoted by SEQ. ID.Nos. 4 and 5. Phosphorothioation of inner pyrimidines has been shown toincrease stability and protect from endonuclease cleavage [Pirollo, K.F. et al. (2003) Pharmacology & Therapeutics 99:55-77]. The antisenseoligonucleotides of the invention are partially phosphorothioated, andare thus less toxic than antisense oligonucleotides that havephosphorothioate modifications in all nucleotides.

Nevertheless, further modifications, like 2-O-methylation, may be foundin the first three and/or the last three nucleotides of saidoligonucleotide [EP 260,032].

As shown in Examples 5 to 7, the antisense oligonucleotides of theinvention may be used as inhibitors of inflammation processes related tocPLA₂ expression and activity.

The antisense oligonucleotide of the invention is thus suitable for usein the treatment and/or prevention of any one of rheumatoid arthritis,ARDS, asthma, rhinitis, idiopathic pulmonary fibrosis, peritonitis,cardiovascular inflammation, myocardial ischemia, reperfusion injury,atherosclerosis, sepsis, trauma, diabetes type II, retinopathy,psoriasis, gastrointestinal inflammation, cirrhosis and inflammatorybowel disease, neurodegenerative diseases such as AD, PD and ALS, aswell as brain ischemic and traumatic injury, i.e. in all diseases whereoxidative stress has a significant role in its pathogenesis, and inwhich there is accelerated release of eicosanoids and superoxides byreactive microglia.

Although a study in human monocytes demonstrated that inhibition ofcPLA₂ expression also inhibits NADPH oxidase activity [Li, Q. andCathcart, M. K. (1997) id ibid.], another report had shown that residentperitoneal macrophages from cPLA₂-deficient mice can, under normalstimulation, release superoxide [Gijon, M. A. et al. (2000) J. Biol.Chem. 275: 20146]. In contrast, the present inventors have nowdemonstrated that the antisense oligonucleotides, which were efficientin inhibiting cPLA₂ expression in mice macrophages, were also efficientin inhibiting superoxide production in macrophages, as well as inmonocytes, neutrophils and microglia cells. This disparity may beexplained by the fact that often “knockout” animal models have normalphenotypes due to, for example, over expression and compensation ofisoenzymes, and thus do not accurately mirror the effects of the lack ofthe gene (i.e., the protein or enzyme) in study. Most importantly, thepresent results were obtained using low levels of oligonucleotides of 1μM (final concentration). It is important that the present antisenseoligonucleotides are effective at low, non-toxic concentrations, whichmakes them suitable for use in clinical purposes, i.e., as a therapeuticagent for the treatment of conditions where inhibition of cPLA₂ isdesirable, as discussed herein.

In addition, the antisense oligonucleotides of the invention aresuitable for use in the treatment and/or prevention of conditions inwhich microglia cells are activated, for example by LPS, and releasereactive oxygen species (ROS) and/or pro-inflammatory mediators, forexample. Said conditions are selected from the group consisting ofinflammations, infections, and ischemic disease.

The antisense oligonucleotide of the invention may also be used forinhibiting superoxide production and release. Usually, said inhibitionis effectuated in neutrophils, monocytes and macrophages, preferably inneutrophils.

Activated neutrophils are the first cells arriving at the inflammationsite, which then release high levels of eicosanoid and superoxides whichaccelerates the inflammatory process. Consequently, neutrophils aredirect effectors of the pathogenesis of the various inflammatorydiseases, and directly inhibiting their function is an efficient way toreduce inflammatory processes. Monocytes (and macrophages) are thesecond cell population to arrive at the site of inflammation, and thustheir inhibition is also important to stall inflammation.

In addition, the antisense oligonucleotides of the invention may be usedfor inhibiting eicosanoid and ROS production released by microglia thatare induced by amyloid β (Aβ) plaque formation or by other agents suchas LPS or cytokines. ROS formation has been shown to be responsible forthe dysfunction or death of neuronal cells that contributes to thepathogenesis of various neurological diseases.

By “analogs and derivatives” is meant the “fragments”, “variants”,“analogs” or “derivatives” of said nucleic acid molecule. A “fragment”of a molecule, such as any of the oligonucleotide sequences of thepresent invention, is meant to refer to any nucleotide subset of themolecule. A “variant” of such molecule is meant to refer a naturallyoccurring molecule substantially similar to either the entire moleculeor a fragment thereof. An “analog” of a molecule can be withoutlimitation a paralogous or orthologous molecule, e.g. a homologousmolecule from the same species or from different species, respectively,i.e., an antisense oligonucleotide complementary to the equivalentregion of the gene in a different species, which therefore may haveslight changes in the sequence.

Further derivatives of the antisense oligonucleotides of the inventionare those labeled or conjugated to a reporter molecule, such that theantisense oligonucleotide of the invention may be traced and/or detectedin the organism. Any label or reporter molecule that allow its detectionmay be suitable, like e.g. biotin, fluorescein, rhodamine,4-(4′-Dimethylamino-phenylazo)benzoic acid (“Dabcyl”);4-(4′-Dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)(“Dabsyl”); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid(“EDANS”); Psoralene derivatives, haptens, cyanines, acridines,fluorescent rhodol derivatives, cholesterol derivatives, radioactivelabels, as well as metal particles (e.g. gold).

In a second aspect, the present invention relates to a pharmaceuticalcomposition comprising as active agent at least one antisenseoligonucleotide as defined in the invention, or functional analogs,derivatives or fragments thereof.

Thus, the antisense oligonucleotide of the invention is generallyprovided in the form of pharmaceutical compositions. Said compositionsare for use by injection, topical administration, or oral uptake.

Alternatively, the pharmaceutical composition of the invention maycomprise as active agent a combination of at least two antisenseoligonucleotides as defined in the invention, or functional analogs,derivatives or fragments thereof. Preferably, said combination comprisesthe following oligonucleotides: SEQ. ID. No.1 together with SEQ. ID.No.3, or SEQ. ID. No.1 together with SEQ. ID. No.2, or SEQ. ID. No.1together with SEQ. ID. No.6, or SEQ. ID. No.1 together with SEQ. ID.No.2 and SEQ. ID. No.3, or SEQ. ID. No.4 together with SEQ. ID. No.6, orSEQ. ID. No.2 together with SEQ. ID. No.6, or SEQ. ID. No.2 togetherwith SEQ. ID. No.3, or SEQ. ID. No.3 together with SEQ. ID. No.6.

In the Examples described herein below, it is clear how both inhibitionof cPLA₂ and inhibition of superoxide production were much moreefficient when the antisense oligonucleotides were used in combinationof two or three together in the same reaction.

In one embodiment, the pharmaceutical composition of the invention isintended for the treatment of inflammation processes related to cPLA₂expression and/or free radical release by phagocyte NADPH oxidase.

In another embodiment, the pharmaceutical composition of the inventionis intended for the treatment of inflammatory conditions, wherein saidinflammatory conditions may be any one of rheumatoid arthritis, ARDS,asthma, rhinitis, idiopathic pulmonary fibrosis, peritonitis,cardiovascular inflammation, myocardial ischemia, reperfusion injury,atherosclerosis, sepsis, trauma, diabetes type II, retinopathy,psoriasis, gastrointestinal inflammation, cirrhosis and inflammatorybowel disease, neurodegenerative diseases such as AD, PD and ALS, aswell as brain ischemic and traumatic injury, i.e. in all diseases whereoxidative stress has a significant role in its pathogenesis, and inwhich there is accelerated release of eicosanoids and superoxides byreactive microglia.

In a further embodiment, the pharmaceutical composition of the inventionis intended for the treatment of conditions related to Aβ plaqueaccumulation. Said conditions are generally neurodegenerative diseases,preferably Alzheimer's, Parkinson's, ALS, or brain ischemic andtraumatic injury.

The pharmaceutical composition of the invention is also intended to beused in the treatment and/or prevention of conditions, for exampleexposure to LPS, in which microglia cells are activated and release ROSand/or pro-inflammatory mediators (eicosanoid). Said conditions areselected from the group consisting of inflammations, infections, andischemic disease.

Preferred uses of the pharmaceutical compositions of the invention byinjection are subcutaneous injection, intraperitoneal injection,intravenous and intramuscular injection.

The pharmaceutical composition of the invention generally furthercomprises a diluent, and/or a buffering agent, i.e. an agent whichadjusts the osmolarity thereof, and optionally, one or more carriers,stabilizers, excipients and/or additives as known in the art, e.g., forthe purposes of adding flavors, colors, lubrication, or the like to thepharmaceutical composition.

A preferred buffering agent is Tris, consisting of 10 mM Tris, pH7.5-8.0, which solution is also adjusted for osmolarity.

For in vivo use, the antisense oligonucleotides are suspended is steriledistilled water or in sterile saline.

Carriers may include starch and derivatives thereof, cellulose andderivatives thereof, e.g., microcrystalline cellulose, xantham gum, andthe like. Lubricants may include hydrogenated castor oil and the like.

The preparation of pharmaceutical compositions is well known in the artand has been described in many articles and textbooks, see e.g.,Remington's Pharmaceutical Sciences, Gennaro A. R. ed., Mack PublishingCo., Easton, Pa., 1990, and especially pp. 1521-1712 therein.

Pharmaceutical compositions for topical administration may includetransdermal patches, ointments, lotions, creams, gels, drops,suppositories, sprays, liquids and powders. Conventional pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

The pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical compositions of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product. Such compositions may be formulated into any of manypossible dosage forms such as, but not limited to, tablets, capsules,liquid syrups, soft gels, suppositories, and enemas. The compositions ofthe present invention may also be formulated as suspensions in aqueous,non-aqueous or mixed media. Aqueous suspensions may further containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

The pharmaceutical composition of the invention may further compriseother active agents, e.g. antibiotics, analgesics and the like.

In another aspect, the present invention provides the use of theantisense oligonucleotide as defined in the invention, for thepreparation of a pharmaceutical composition for the treatment and/orprevention of inflammatory conditions, wherein said inflammatoryconditions may be any one of rheumatoid arthritis, ARDS, asthma,rhinitis, idiopathic pulmonary fibrosis, peritonitis, cardiovascularinflammation, myocardial ischemia, reperfusion injury, atherosclerosis,sepsis, trauma, diabetes type II, retinopathy, psoriasis,gastrointestinal inflammation, cirrhosis and inflammatory bowel diseaseneurodegenerative diseases such as AD, PD, and ALS, as well as brainischemic and traumatic injury, i.e. in all diseases where oxidativestress has a significant role in its pathogenesis, and in which there isaccelerated release of eicosanoids and superoxides by reactivemicroglia.

Furthermore, the present invention provides the use of an antisenseoligonucleotide as defined in the invention for the treatment ofconditions associated with cPLA₂ activation, as well as for thetreatment and/or prevention of conditions related to Aβ plaqueaccumulation. Generally said conditions are neurodegenerative diseases,selected from the group consisting of Alzheimer's disease, Parkinson'sdisease, ALS, and brain ischemic and traumatic injury.

Hence, the present invention presents a method of treatment ofconditions associated with cPLA₂ activation, comprising administering atherapeutically effective amount of at least one antisenseoligonucleotide as defined in the invention, or compositions comprisingthereof, to a subject in need.

Said therapeutic effective amount, or dosing, is dependent on severityand responsiveness of the disease state to be treated, with the courseof treatment lasting from several days to several months, or until acure is effected or a diminution of the disease state is achieved.Optimal dosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. Persons of ordinary skill caneasily determine optimum dosages, dosing methodologies and repetitionrates. Optimum dosages may vary depending on the relative potency ofindividual oligonucleotides, and can generally be estimated based onEC₅₀, found to be effective in in vitro as well as in in vivo animalmodels. In general, dosage is from 0.01 μg to 10 mg per kg of bodyweight, and may be given once or more daily, weekly, monthly or yearly,or even once every 2 to 20 years. Persons of ordinary skill in the artcan easily estimate repetition rates for dosing based on measuredresidence times and concentrations of the antisense oligonucleotide inbodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 10 mg per kgof body weight, once or more daily.

As demonstrated in the following Examples 5 to 7, optimal dosage usedfor treatment of the inflammatory conditions is 1-2 mg/kg/day givendaily for between 5 up to 14 days (Example 5), or given in one or twodoses of 1-2 mg/kg/day after inflammation (Examples 6 and 7).

The use of antisense oligonucleotides for inhibiting cPLA₂ expression isan innovative treatment for local inflammatory diseases, since it willinhibit specifically the elevated cPLA₂ at the site of inflammation andwill not affect normal cPLA₂ expression. This treatment is potentiallyeven more effective when affecting also the activity of activated orprimed phagocytes involved in the pathogenesis of such diseases, sincethese cells secrete high levels of eicosanoids and superoxides, whichaccelerate the inflammation process.

Therefore, the present invention also provides a method of treatment ofinflammatory conditions, wherein said inflammatory conditions are anyone of rheumatoid arthritis, ARDS, asthma, rhinitis, idiopathicpulmonary fibrosis, peritonitis, cardiovascular inflammation, myocardialischemia, reperfusion injury, atherosclerosis, sepsis, trauma, diabetestype II, retinopathy, psoriasis, gastrointestinal inflammation,cirrhosis and inflammatory bowel disease, neurodegenerative diseasessuch as AD, PD and ALS, as well as brain ischemic and traumatic injury,i.e. in all diseases where oxidative stress has a significant role inits pathogenesis, and in which there is accelerated release ofeicosanoids and superoxides by reactive microglia, comprisingadministering a therapeutically effective amount of at least oneantisense oligonucleotide as defined in the invention, or compositionscomprising thereof, to a subject in need.

In addition, involvement of cPLA₂ in the regulation of superoxideproduction by NADPH oxidase in microglia cells suggests that cPLA₂antisense oligonucleotides may be used in the treatment ofneurodegenerative diseases, selected from the group consisting ofAlzheimer's disease, Parkinson's disease, ALS, and brain ischemic andtraumatic injury.

Likewise, the present invention also provides a method for the treatmentand/or prevention of conditions in which microglia cells are activated,exposed to LPS for example, and release ROS and/or pro-inflammatorymediators, and wherein aid conditions are selected from the groupconsisting of inflammations, infections, and ischemic disease. Saidmethod comprises administering a therapeutically effective amount of atleast one antisense oligonucleotide as defined in the invention, orcompositions comprising thereof, to a subject in need.

As a final aspect, the present invention provides a method of treatmentof neurodegenerative diseases, as well as brain damage (caused by strokeor trauma, for example), comprising administering a therapeuticallyeffective amount of at least one antisense oligonucleotide as defined inthe invention, or compositions comprising thereof, to a subject in need.Said conditions are generally neurodegenerative diseases, preferablyAlzheimer's and Parkinson's disease, or brain ischemic and traumaticinjury, in which there is accelerated release of eicosanoid andsuperoxides by reactive microglia.

Various methods of administration may be used for delivering theantisense oligonucleotide of the invention to a subject in need.Oligonucleotides may be delivered via intravenous (i.v.), intramuscular(i.m.) intraperitoneal (i.p.) injections, orally (in liquid form orprepared as dosage unit forms like capsules, pills, lozenges, etc.). Inorder to be effective therapeutically, oligonucleotides should beprepared in a way that would enable their stability in the systemfollowing injection, or yet more preferably, following oraladministration. Alternatively, the oligonucleotides of the invention mayalso be delivered via transdermal delivery using patches, ointment orcream.

In addition, pharmaceutical compositions comprising as active agent theantisense oligonucleotides described in the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

As shown in the following Examples, preliminary trials with threedifferent models of inflammation in mice and rats (arthritis, ARDS andperitonitis), demonstrate the effectiveness of the specific cPLA₂antisense oligonucleotides as anti-inflammatory treatment.

The present invention is defined by the claims, the contents of whichare to be read as included within the disclosure of the specification.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, process steps, and materialsdisclosed herein as such process steps and materials may vary somewhat.It is also to be understood that the terminology used herein is used forthe purpose of describing particular embodiments only and not intendedto be limiting since the scope of the present invention will be limitedonly by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The following Examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the intended scope ofthe invention.

EXAMPLES Experimental Procedures General Methods of Molecular Biology

A number of methods of the molecular biology art are not detailedherein, as they are well known to the person of skill in the art. Suchmethods include PCR, expression of cDNAs, transfection of mammaliancells, and the like. Textbooks describing such methods are, e.g.,Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, ISBN: 0879693096; F. M. Ausubel (1988) CurrentProtocols in Molecular Biology, ISBN: 047150338X, John Wiley & Sons,Inc. Furthermore, a number of immunological techniques are not in eachinstance described herein in detail, like for example Western Blot, asthey are well known to the person of skill in the art. See, e.g., Harlowand Lane (1988) Antibodies: a laboratory manual. Cold Spring HarborLaboratory.

Oligonucleotides

TABLE 1 Oligonucleotides used in the following examples SequenceSequence  Oligon. 5'-3' Reference ID. No. C2 ttcaaaggtc — SEQ. ID. tcattccaca No. 1 C3 cactataatg — SEQ. ID.  tgctggtaag No. 2 C4caaaacatttt — SEQ. ID.  cctgattagg No. 3 C8 cacagggttt — SEQ. ID. atgtcattat No. 4 C9 ccgtaaactt — SEQ. ID.  gtgggaatac No. 5 C10gctgtcaggg — SEQ. ID.  gttgtag No. 6 P1 gtaaggatcta [Roshak et al. SEQ. ID.  taaatgacat (1994) id ibid;  No. 8 Muthalif et al. (1996) id ibid;  Marshall et al  (1997) id ibid;  Anderson et al. (1997) id ibid.] P2 ccccctttgt [Li and Cathcart SEQ. ID.  cactttggtg(1997) id ibid] No. 9 IS1 gcccaaaact [U.S. Pat. No. SEQ. ID.  ctgttgaa6,008,344] No. 10 IS2 ttgtgaacca [U.S. Pat. No.   SEQ. ID.  gaaacgcc6,008,344] No. 11 Note: the underline shows phosphorothioatednucleotides.

Example 1 Synthesis of Anti-cPLA₂ Antisense Oligonucleotides

Fourteen different partially phosphorothioated [Stein et al. (1988)Nucleic Acids Res. 16:3209-3221] oligonucleotides against cPLA₂ wereoriginally synthesized. Prior to use, the oligonucleotides were purifiedby HPLC and tested for purity by mass spectrometry (Sigma, UK). Prior toselection, the sequences were analyzed by screening for uniqueness usingthe Blast program and were also tested for lack of secondary structureand oligo pairing using Mulfold [Jaeger, J. A. (1989) Methods Enzymol.183: 281-306].

Preliminary experiments (not shown) demonstrated that from thosefourteen, only six oligonucleotides were found to be efficient ininhibiting cPLA₂ expression, and their sequences are detailed in Table 1above.

The fact that only six antisense oligonucleotides displayed significantactivity suggested that these have increased specificity for the targetsequence. The remaining eight oligonucleotides were used in theexperiments as controls.

The oligonucleotides carried phosphorothioate modifications on the last3 bases at both 5′ and 3′ ends (as indicated by underline, Table 1), andwere engineered using computer based approach using RNADraw V1.1 [MazuraMultimedia, Sweden] for the first 400 hundred base pairs (N-terminal) ofcPLA₂ mRNA (Table 1).

The partially phosphorothioated oligonucleotides (PPS) are less toxicand more specific than the phosphorothioated oligonucleotides, butsimilar in their stability and cellular uptake. Some PPS contain GGGG(C10) and GGG (C8 and C9) which have been shown to increase cellularuptake (see Table 1). PPS C8, C9 also contain phosphorothioated on innerpyrimidines (t) which have been shown to increase stability and protectfrom endonuclease cleavage (Table 1, underlined). Five of the antisenseoligonucleotides did not contain CpG, which have been shown to stimulateimmune responses. In all of the experiments presented herein, naked PPSwere added since in this form they can be used for in vivo treatment[Pirollo, K. F. et al. (2003) id ibid.], in contrast to the publishedstudies in vitro, which used different delivery systems to increase theantisense uptake. Hence, all the clinical trials with antisenseoligonucleotides are carried out with naked oligonucleotides. Forexample, an in vivo clinical trial did not require cationic lipids foroligonucleotide delivery against Bcl-2 in cancer patients, in contrastto the experiments performed in tissue culture [Jansen, B. et al. (2000)Lancet 356: 1728-1733]. This is usually the case in in vivo assays, andto date it is not well understood why a vector does not appear to benecessary. One hypothesis is that the oligonucleotides interact withcirculating proteins, which both protect them against degradation, andserve as carriers in ways not yet understood [Dias, N. and Stein, C. A.(2002) Eur. J. Pharmac and Biopharmac. 54: 263-269].

As shown in FIGS. 3-7, there is a clear correlation between inhibitionof cPLA₂ expression by the PPS antisense oligonucleotides and stimulatedproduction of superoxides. Each PPS oligonucleotide caused significantinhibition of cPLA₂ protein expression in different phagocytic celltypes, and their combinations caused significantly improved inhibition.

As shown in FIGS. 3 and 4, the PPS antisense oligonucleotides of theinvention are far more efficient than those previously reported [U.S.Pat. No. 6,008,334; Roshak et al. (1994) id ibid; Muthalif et al. (1996)id ibid; Marshall et al (1997) id ibid; Anderson et al. (1997) id ibid;Li, Q. and Cathcart, M. K. (1997) id ibid.; Zhao, X. et al. (2002) idibid.]. Moreover, in the previous reports, these antisenseoligonucleotides were tested only for their capacity to inhibit mRNAsynthesis [U.S. Pat. No. 6,008,334].

The effect of the number (and kind) of modifications at the two ends ofthe oligonucleotides was also evaluated (data not shown). The rank orderof the efficiency in inhibiting cPLA₂ expression and NADPH oxidaseobserved was as follows: PPS with 3 modifications>PPS with 2modifications>PPS with 1 modification>no modification.

The PPS antisense oligonucleotides were more efficient in inhibitingcPLA₂ protein expression in the phagocytic cells, as shown formonocytes, than endothelial or epithelial cells (data not shown). Thisphenomenon has advantages especially regarding treatment of inflammationdirectly affecting the phagocyte at the site of inflammation, withoutaffecting the organ.

Example 2 Effect of cPLA₂ Antisense Oligonucleotides on cPLA₂ Expressionand Superoxide Production in Peripheral Blood Human Monocytes

The expression of cPLA₂ protein was analyzed by Western blot analysisusing antibodies against cPLA₂ that were raised by the inventors andwhich are much more efficient than those available on the commercialmarket (FIG. 2).

The effect of the different PPS antisense oligonucleotides and theircombinations on cPLA₂ expression and on superoxide production wasstudied in peripheral blood human monocytes (FIGS. 3 and 4) and inmurine macrophages (FIG. 5). Naked (free of transfection solutions likelipofectin, etc.) PPS antisense oligonucleotides (final concentration of1 μM) were added to the cell suspension in RPMI containing 10% FCS for16 h at 37° C. The same cells were analyzed for superoxide productionstimulated with 5 ng/ml PMA (by cytochrome c reduction) and for theexpression of cPLA₂ in cell lysates (by Western blot analysis). Asshown, each of the PPS antisenses caused between 50-75% inhibition ofsuperoxide production, which is in correlation with their effect oninhibiting cPLA₂ expression. The level of cPLA₂ was quantitated bydensitometry in a reflectance mode (Hoefer, Hoefer ScientificInstruments, San Francisco, USA). The correlation between these twoparameters is demonstrated in FIG. 6.

As shown in FIGS. 3 and 4, respectively, P1 [Roshak et al. (1994) idibid; Muthalif et al. (1996) id ibid; Marshall et al (1997) id ibid;Anderson et al. (1997) id ibid] and P2 [Li, Q. and Cathcart, M. K.(1997) id ibid.], and IS1 and IS2 [U.S. Pat. No. 6,008,334] did not haveany effect either on cPLA₂ expression or on superoxide production. Mostimportantly, oligonucleotides IS1 and IS2, were reported, in U.S. Pat.No. 6,008,334, to cause 100% and 92% inhibition of mRNA expression,respectively. However, their effect was analyzed only by inhibition ofmRNA expression and not by inhibition of cPLA₂ protein expression.

In their original publications, P1, P2 IS1 and IS2 were synthesized withphosphorothioate modifications in all bases. The present inventor foundthat, when fully phosphorothioated, these oligonucleotides were toxic tothe cells, and caused the killing of about 60-70% of the cells after 16hours of incubation (data not shown). These results are consistent witha previous report showing that oligonucleotides with phosphorothioatemodifications in all bases are much more toxic [Pirollo, K. F. et al.(2003) id ibid.]. Thus, it is important to note that, for this study,P1, P2, IS1 and IS2, differently from the original oligonucleotides,were synthesized with phosphorothioate modifications only in the firstand last three bases, in order to more accurately compare their effectto the effect of the PPS antisense oligonucleotides synthesized by thepresent inventor.

The results shown in FIGS. 2 and 3 demonstrate that the antisenseoligonucleotides synthesized (and claimed) by the inventor are superiorto the oligonucleotides described in the literature. In particular, itmay be highlighted that the former are:

a. Less toxic;b. Much more potent in inhibiting cPLA₂ protein expression.

The antisense oligonucleotides are especially preferred, compared to P1,P2, IS1 and IS2 since they may be introduced into the cells and performtheir activity without cell delivery systems like lipofectin.

Example 3 Effect of cPLA₂ Antisense Oligonucleotides on cPLA₂ Expressionand Superoxide Production in Peripheral Blood Human Neutrophils

The effect of the different PPS antisense oligonucleotides, and theircombinations, on cPLA₂ expression and on superoxide production wasstudied in peripheral blood human neutrophils. Naked antisenseoligonucleotides were added (at 1 μM final concentration) to 2×10⁵neutrophils for 6 h at 37° C. Neutrophils (95% purity) were isolated byFicoll/Hypaque centrifugation, dextran sedimentation and hypotonic lysisof erythrocytes [Levy, R. et al. (2000) Blood 95:660-665]. As shown inFIG. 7, there was a slight though significant (P<0.05) inhibition ofsuperoxide production even after 6 hours of incubation with theneutrophils. In another experiment, a similar effect was shown after 16h of incubation (data not shown).

Inhibition of superoxide production by the various oligonucleotideantisenses was significantly improved when the cells were stimulated byphysiological agonists such as fMLP, opsonized zymosan (OZ) (FIG. 8),LTB4, angiotensin II or AGEs (advanced glycation end products) (data notshown) which bind specific receptors on cell membranes.

The effect of the PPS was significantly higher when neutrophils werepurified from patients with inflammatory diseases (like rheumaticarthritis, asthma or sepsis, data not shown), than when the neutrophilswere from healthy controls. This phenomena is consistent with theinventor's earlier study [Levy, R. et al. (2000) id ibid.] whichreported that the level of cPLA₂ in neutrophils is higher during thediseases indicating increase rate of synthesis, and thus more prone totargeting.

It is important to note that the experiments portrayed in FIGS. 3 to 6were performed with the non-physiological stimulant PMA, an extremelypotent activator of NADPH oxidase that bypasses the receptors and actson PKC. This enabled the analysis of the role of the antisenses directlyon NADPH oxidase, since under these conditions the effect of theexpression of the different receptors and their binding to stimulants iseliminated.

Example 4 Effect of cPLA₂ Antisense Oligonucleotides on cPLA₂ Expressionand Superoxide Production in Rat Microglia

The effect of the antisenses and their combinations was studied onmicroglia isolated from rat brain. The PPS antisense oligonucleotideswere added (at 1 μM final concentration) to microglia cells for 16 h(similar to the conditions used for monocytes). There was a significantinhibition of cPLA₂ protein expression. The cells were stimulated with 2mg/ml PMA or with 10 μM of Amyloid (3, and superoxide production wasanalyzed by the fluorescence probe Amplex Red. As shown in FIG. 9, theantisenses caused significant inhibition of superoxide production, incorrelation with cPLA₂ protein expression. Interestingly, antisense C8,which did not match the rat sequence by 1 base, did not cause inhibitionof superoxide production, indicating the high specificity of theantisenses. Inhibition was much higher when the cells were stimulatedwith Amyloid β. Incubation of microglia for 48 h with the antisenseoligonucleotides caused higher inhibition of cPLA₂ expression and ofsuperoxide production (data not shown). The results are of particularimportance since Amyloid β plays an important role in the pathogenesisof brain and neurodegenerative diseases, such as Alzheimer.

Incubation of all cell types with X150 antisense concentration was nottoxic to the cells and did not affect cell functions which are notregulated by cPLA₂ (data not shown).

Example 5 Animal Model of Inflammation: Collagen-Induced Arthritis

Collagen-induced arthritis (CIA) is an experimental model of autoimmunearthritis that has many clinical and pathological similarities torheumatoid arthritis (RA). CIA is induced by immunizing susceptibleanimals (e.g. DBA black mice) with type II collagen (CII) as described[Bendele, A. M. et al. (2000) Arthritis & Rheumatism, 43:2648-58]. Allmice were maintained in a specific pathogen-free environment, and fedstandard mouse chow and water. Chick CII (Sigma-Aldrich, 2 mg/ml) wasdissolved overnight at 4° C. in 10 mM acetic acid and combined with anequal volume of CFA (complete Freund's adjuvant). CFA was prepared bymixing 100 mg of heat-killed M. tuberculosis (H37Ra, Difco, Detroit,Mich., USA) with 20 ml of incomplete Freund's adjuvant (Sigma-Aldrich,St. Louis, Mo., USA). Mice (aged 7 to 10 weeks) were injectedintradermally at the base of the tail and boosted at day 7 or 21.Control mice were treated with CFA without CII. The severity ofarthritis was monitored by direct examination with a digital caliperaccording to the following scale: grade 0, no swelling; 1, slightswelling and erythema; 2, pronounced inflammation; and 3, jointrigidity. Each limb was graded, giving a maximum possible score of 12per animal. Fluid from animal paws was aspirated to determine cytokines(mRNA and protein level) and white blood cells. After sacrifice, pawswere collected, fixed, decalcified, and paraffin embedded. Sections werestained with hematoxylin and eosin and scored according to the followingscale: 0, no inflammation; 1, slight thickening of the synovial celllayer and/or some inflammatory cells in the sublining; 2, thickening ofthe synovial lining, infiltration of sublining, and localized cartilageerosions; and 3, infiltration in the synovial space, pannus formation,cartilage destruction, and bone erosion.

Detection of Anti-CII Antibodies:

ELISA for antibodies to CII was performed by HRP-conjugated secondaryantibody specific for IgG1, IgG2a, IgG2b, IgG3, or IgM and IgA.

Cell Isolation:

Positive selection of splenocyte subsets of synovial cells wereperformed using specific biotin-conjugated antibodies against CD11b,CD3, CD4, and CD8, and biotin binder Dynabeads. After positive selectionof CD11b⁺ and CD3⁺ synovial cells, synovial cells were incubated withbiotin CD45 and negatively selected using an excess of avidin-magneticbeads. The remaining CD45-negative cells were used for RNA extraction.Synovial fluid leukocytes were obtained from synovial effusions andpurified by Ficoll-Hypaque density gradient centrifugation.

The model of CIA was successfully developed by the inventors, asdemonstrated, e.g. by the swollen limb of an exacerbated CIA mousecompared to the limb of a control mouse (FIG. 10A). Histologicalassessment of the CIA mouse limb (FIG. 10B) was in correlation with theseverity of arthritis, monitored by direct examination of the limb aspresented in FIG. 10A. Infiltration of inflammatory cells (especiallyneutrophils) is shown in a joint section of a CIA mouse (FIG. 10C). CIAmice showed severe difficulties in walking (data not shown).

Based on preliminary experiments performed with various PPSconcentrations and combinations, the optimal concentration of 2 mg/Kgwas defined, for a combination of 3 different PPS (2, 4 and 10), alsoreferred to herein as cocktail. This concentration is within the rangeused for human therapy and in animal models.

For treatment of the inflammatory condition, sterile stock of antisenses(100 μM) is dissolved in sterile saline at the desired concentrations,and injected to sick mice either i.v., intradermally at the base of thetail, or at the inflamed joints.

As shown in FIG. 11, intravenous injection of 2 mg/kg of the “cocktail”every day for 14 days caused significant remission of the arthritis, asdetected by a reduction in swelling of the limb (FIG. 11A), by diseaseseverity score, by full recovery of the mice ability to move and to runfreely (data not shown), and by reduction in serum IL-6 and TNFα levels(FIG. 11B).

Example 6 Animal Model of Inflammation: Peritonitis

Model mice peritonitis is being developed in CD1 mice by injection ofCandia as previously described [Levy, R. et al. (1989) J. Biol. Regul.Homeost. R Agents 3:5-12], or by injection of different doses of grampositive bacteria peritonitis will be induced with lethal doses ofcandida or bacteria (S. epidermitis) or gram negative (E. Coli) whichcause animal killing and with moderate doses which cause a moderatedisease. The 50% lethal doses were determined as 6×10⁸ CFU for S.epidermits and 1.5×10⁷ for CFU E. coli. The markers for infection and/orinflammation are: the number and population of blood cells in theperitonitis area, the concentration of inflammatory cytokines in theblood, such as TNFα, IL1 and IL6, and histological analyses ofperitoneal sections. Mice injected with a lethal dose of bacteria aretreated with antisenses 2 h after injection of the bacteria followed byantisenses injection in different time intervals. In these experiments,the effect of antisenses on mice survival is evaluated. For miceinjected with lower doses of bacteria, the effect of antisenses(injected as described) is evaluated on the pathology by the infectionand/or inflammation markers.

Example 7 Animal Model of Inflammation: Sterile Peritonitis

A model of sterile peritonitis was developed in ICR mice byintraperitoneal injection of 3 ml sterile 4% thioglycollate (TG) asdescribed previously [Segal, B. H. et al. (2002) J Leukoc Biol 71:410].Assessment of inflammation was determined by the number and populationof blood cells in the peritonitis cavity, the concentration of LTB₄ andthe presence of inflammatory cytokines, TNFα and IL6, in cell freeperitoneal fluid and in serum. 10 ml of sterile PBS were injected to theperitoneal cavity in order to collect the cells. The number of cells wasdetermined by microscopy after trypan blue staining. The composition ofthe cell population was determined by FACS using antibodies against antimouse neutrophils (MCA771F), anti mouse macrophages (F4/80) and antimouse lymphocytes (CD3). The composition of the cell population was alsodetermined under microscopy after Giemsa staining. FIGS. 12A-12B presentthe changes in white blood cell count (FIG. 12A) and cell population(FIG. 12B) during 4 days of sterile peritonitis. High levels ofneutrophils were detected 24 h after TG injection, which were laterreplaced by monocyte-macrophages. There was a significant elevation instimulated phagocyte superoxide production, with the highest rate at 24h post peritonitis induction.

The levels of LTB4 in the serum and in the peritoneal cavity during 24 hof sterile peritonitis are demonstrated in FIGS. 13A-13B. 1 hour afterthe induction of peritonitis there was a significant elevation of LTB4levels in the blood. LTB4 levels stayed high during 5 h and thandecreased to half after 8 hours. At 16 h after peritonitis induction,LTB4 could not be detected in the blood. A similar pattern was observedin the peritoneal cavity, but the decay was slower and only after 24 h,LTB4 ceased to be detected.

According to the present results and consistent with the literature, thefirst cells to accumulate in the peritonitis model were neutrophils.Thus, in the first set of experiments the effect of the antisenseoligonucleotides was analyzed primarily on neutrophil accumulation andactivity, after 24 h of induction of sterile peritonitis. Two doses of aspecific combination of PPS antisense oligonucleotides against cPLA₂(the “cocktail”) dissolved in sterile water (0.2 ml) were administeredi.v. in the tail of the mice 1 h and 4 h after induction of peritonitis.As demonstrated in FIGS. 14A-14C (results of two mice) this treatmentcaused a significant reduction in the number of neutrophils present inthe peritoneal cavity (represented as cell number and cell populationdistribution detected by FACS analysis) and a slight inhibition ofstimulated superoxide production by the peritoneal cells 24 h afterinduction of inflammation. The efficiency of the cocktail on reductionof peritoneal recruited neutrophils and on superoxide production in 34mice compared to 34 untreated mice with sterile peritonitis is presentedin FIGS. 15A-15B. Superoxide release from unstimualted cells is of greatimportance since it reflects the behavior of the peritoneal cells in theperitoneum cavity. Thus, the release of superoxide from resting cellswas assessed with the fluorescent probe Dihydrorhodamine-123 (1 mM)which is a very sensitive method and can detect low levels ofsuperoxide. As shown in the representative results in FIG. 16, thecocktail treatment for 24 h after induction of peritonitis significantlydecreased the release of superoxides by peritoneal cells.

The effect of the antisense oligonucleotide treatment was studied onperitoneal cell population during 4 days of sterile peritonitis. Thenumber of recruited neutrophils was dramatically reduced and that ofmacrophages was significantly lower in the peritoneum of treated miceduring the 4 days of peritonitis (FIG. 17A-17B). Stimulated superoxideproduction by the peritoneal cells of the antisense-treated mice wassignificantly lower than in the untreated mice during the four days ofperitonitis (FIG. 18A-18B).

The antisense cocktail treatment reduced the levels of LTB₄ in theperitoneal cavity as measured during 24 h after induction of peritonitis(FIGS. 19A-19B) in correlation with the reduction of neutrophilsrecruited to peritoneal cavity (FIGS. 20A-20B).

Accumulation of antisense oligonucleotides in peritoneal blood cells 24h after i.v. injection was detected using fluorescently labeledantisense oligonucleotides (labeled with FITC at the last nucleotide)(FIGS. 21A-21B). Distribution of the fluorescent antisenseoligonucleotides in the different organs by histological section wasalso analyzed (data not shown). In further experiments, differentantisense overdoses were administered, in order to determine theirtoxicity. A cocktail X100 was not toxic to the mice (data not shown).

Example 8 Animal Model of Inflammation: Experimental Acute Lung InjuryInduced by 1—LPS/Zymosan Administration.

Mice are anesthetized and mechanically ventilated. During theexperiments, oxygen gas is supplied continuously to the ventilatorysystem. One minute prior to intravenous (i.v.) administration, two deepinhalations (3× tidal volume) are delivered to standardize volumehistory and measurements are made as baseline. Mice then receive 3 mg/kgof LPS from Escherichia coli O111:B4 (Sigma Chemical Co., St. Louis,Mo.) i.v. Two hours later, 10 mg/kg of zymosan A from Saccharomycescerevisiae (Sigma) are administered i.v. In saline-treated group,animals receive saline instead of LPS and zymosan in the same manner,and serve as controls. In all groups, measurements are made at 30 minuteintervals for 4 hours. In some animals, the observation period extendedup to 6 hours. To assess the development of lung injury physiologically,EL (a reciprocal of lung compliance) is measured. Tracheal pressure(Ptr), flow and volume (V) will be measured. EL and lung resistance (RL,data not shown) are calculated by adjusting the equation of motion:Ptr=ELV+RL(dV/dt)+K, where K is a constant. Changes in EL reflect lungparenchymal alterations and stiffening of the lungs.

2—HCl Aspiration.

After baseline measurements, anesthetized and mechanically ventilatedmice receive 2 ml/kg of HCl (pH=1.5) i.t., followed by a bolus of air(30 ml/kg). In saline-treated group, animals receive saline instead ofHCl in the same manner and serve as controls. In all groups measurementsare made at 30 minute intervals for 2 hours. In some animals, theobservation period is up to 5 hours. EL measurement is a physiologicparameter to assess acute lung injury.

Assessment of Pulmonary Edema—At the end of experiment, the lung wet/drymass ratios are calculated to assess pulmonary edema. After the trappedblood is drained from the excised lungs, measurements of the lung wetmass are made. The lungs are then heated at 90° C. to constant mass in agravity convection oven for 72 h and the residue weighed as the lung drymass.

Bronchoalveolar Lavage Fluid—At the end of experiment, bronchoalveolarlavage (BAL) is performed (using 5×1 ml phosphate-buffered saline) ineach group. In each animal, 90% (4.5 ml) of the total injected volume isconsistently recovered. After BAL fluid is centrifuged at 450 g for 10minutes, the total and differential cell counts of the BAL fluid isdetermined from the cell fraction. The supernatant is stored at −70° C.until measurement of protein content. The concentration of protein ismeasured by Lowry's method using bovine serum albumin as a standard.

Thromboxane and Leukotriene Measurement—is determined by using enzymeimmunoassay (EIA) kits.

What is claimed is:
 1. A method of treating a condition related to Aβplaque accumulation in a subject, comprising administering to thesubject a therapeutically effective amount of at least one antisenseoligonucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1-6, thereby treating the conditionrelated to Aβ plaque accumulation.
 2. The method of claim 1, whereinsaid condition is selected from the group consisting of Alzheimer'sdisease, Parkinson's disease and amyotrophic lateral sclerosis (ALS). 3.The method of claim 1, wherein said condition is Alzheimer's disease orALS.
 4. The method of claim 1, wherein said at least one antisenseoligonucleotide consists of a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1-6.
 5. The method of claim 1, whereinsaid at least one antisense oligonucleotide comprises an oligonucleotidehaving a nucleic acid sequence as set forth in SEQ ID NO: 1, anoligonucleotide having a nucleic acid sequence as set forth in SEQ IDNO: 3 and an oligonucleotide having a nucleic acid sequence as set forthin SEQ ID NO:
 6. 6. The method of claim 3, wherein said at least oneantisense oligonucleotide comprises an oligonucleotide having a nucleicacid sequence as set forth in SEQ ID NO: 1.